Recent studies have documented that 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) exerts rapid effects on cytosolic and nuclear Ca2+ levels, pH, and phospholipid metabolism. The rapid effects on cytosolic and nuclear Ca2+ also occur in osteoblast-like cell lines with (ROS 17/2.8) and without (ROS 24/1) the classic vitamin D receptor. It is our objective to further define the interrelationships between the hormone, e.g., alterations in gene transcription and response to parathyroid hormone (PTH). The 1alpha,25-(OH)2D3 rapid effects on the mobilization of intracellular Ca2+ stores do not appear to require the classic vitamin D receptor. The hormone-induced increments in Ca2+ occur in cells without the receptor (ROS 24/1) and are inhibited by the inactive epimer 1beta,25-dihydroxyvitamin D3 (1beta,25(OH)2D3), which does not bind to the classic receptor. Intracellular Ca2+, pH, and inositol lipids, as well as 1alpha,25-(OH)2D3, appear to be involved in the regulation of cell and nuclear function. Pretreatment of osteoblast-like cells with 1alpha,25-(OH)2D3 attenuates the response of these cells to PTH. Increments in intracellular Ca2+ alter gene expression. 1alpha,25-(OH)2D3 rapidly and stereospecifically increases cytosolic and nuclear Ca2+ in hepatocytes and osteoblast-like cells. The hormone-induced changes in cell pH and phospholipid metabolism precede the increments in Ca2+ in hepatocytes. These relationships in the osteoblast-like cell lines and hepatoma cells have not been defined. Likewise, the relationship between the rapid changes in Ca2+ and altered gene expression, e.g., osteocalcin and procollagen in ROS cells and c-myc in hepatoma cells, decreased collagen synthesis, and attenuated response to PTH, are unknown. It is the purpose of this proposal to 1) examine the effects of 1alpha,25-(OH)2D3 on cytosolic Ca2+, pH, and phospholipid metabolism in clonal osteosarcoma cells with and without the classic vitamin D receptor and in a human hepatoma cell line; 2) define the effects of 1alpha,25-(OH)2D3 on Ca2+ levels, pH, and phospholipid metabolism in nuclei derived from the osteoblast-like cells and normal and transformed liver cells; 3) correlate the rapid changes in the osteoblast-like cells with altered collagen synthesis and response to PTH; and 4) determine the relationship of the rapid effects of 1alpha,25-(OH)2D3 to the hormone's effects on osteocalcin and procollagen gene expression in ROS 17/2.8 cells and c-myc expression in a human hepatoma cell line.